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Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.

Gascoigne KE, Cheeseman IM - Chromosome Res. (2013)

Bottom Line: We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes.Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar.Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.

ABSTRACT
Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

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Induction of an ectopic kinetochore causes genomic rearrangements. 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown in the absence of IPTG for 3 days and then prepared for FISH analysis using probes against chromosome 3 (pink) and LacO sequence (green). Images show representative metaphase spreads after FISH. Note that nonspecific background signal from chromosome 3 probes is also visible on centromeres. Scale bar shows 10 μm. Insets show examples of chromosome rearrangements. Graph shows quantification of the number of chromosomes containing chromosome 3 sequences. N > 15 cells per treatment
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Fig2: Induction of an ectopic kinetochore causes genomic rearrangements. 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown in the absence of IPTG for 3 days and then prepared for FISH analysis using probes against chromosome 3 (pink) and LacO sequence (green). Images show representative metaphase spreads after FISH. Note that nonspecific background signal from chromosome 3 probes is also visible on centromeres. Scale bar shows 10 μm. Insets show examples of chromosome rearrangements. Graph shows quantification of the number of chromosomes containing chromosome 3 sequences. N > 15 cells per treatment

Mentions: For this study, we stably expressed the GFP-CENP-T-LacI fusion in mouse NIH3T3 cells in which an array of lacO repeats was integrated into chromosome 3 (Soutoglou et al. 2007) (Fig. 1a). 3T3 cells are a non-transformed immortalized fibroblast cell line. The 3T3-lacO parental cell line used in this study displayed contact inhibition and anchorage-dependent growth consistent with the lack of transformation. Although we observed moderate whole chromosome aneuploidy in these cells, we did not observe spontaneous genomic rearrangements, indicating the 3T3 cells are stable at the intra-chromosomal level as has been characterized previously (Leibiger et al. 2013). The 3T3-lacO cell line contains a modal number of three copies of chromosome 3 per cell (Fig. 2). One of these copies consists of two copies of chromosome 3 fused together at their q arms by an array of lac operator repeats, forming a structurally dicentric, but functionally monocentric chromosome (Soutoglou et al. 2007). One centromere on this chromosome is inactive, producing a single structure with an active centromere at one end of the chromosome and an array of lacO repeats at the center (Fig. 2).Fig. 1


Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.

Gascoigne KE, Cheeseman IM - Chromosome Res. (2013)

Induction of an ectopic kinetochore causes genomic rearrangements. 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown in the absence of IPTG for 3 days and then prepared for FISH analysis using probes against chromosome 3 (pink) and LacO sequence (green). Images show representative metaphase spreads after FISH. Note that nonspecific background signal from chromosome 3 probes is also visible on centromeres. Scale bar shows 10 μm. Insets show examples of chromosome rearrangements. Graph shows quantification of the number of chromosomes containing chromosome 3 sequences. N > 15 cells per treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713265&req=5

Fig2: Induction of an ectopic kinetochore causes genomic rearrangements. 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown in the absence of IPTG for 3 days and then prepared for FISH analysis using probes against chromosome 3 (pink) and LacO sequence (green). Images show representative metaphase spreads after FISH. Note that nonspecific background signal from chromosome 3 probes is also visible on centromeres. Scale bar shows 10 μm. Insets show examples of chromosome rearrangements. Graph shows quantification of the number of chromosomes containing chromosome 3 sequences. N > 15 cells per treatment
Mentions: For this study, we stably expressed the GFP-CENP-T-LacI fusion in mouse NIH3T3 cells in which an array of lacO repeats was integrated into chromosome 3 (Soutoglou et al. 2007) (Fig. 1a). 3T3 cells are a non-transformed immortalized fibroblast cell line. The 3T3-lacO parental cell line used in this study displayed contact inhibition and anchorage-dependent growth consistent with the lack of transformation. Although we observed moderate whole chromosome aneuploidy in these cells, we did not observe spontaneous genomic rearrangements, indicating the 3T3 cells are stable at the intra-chromosomal level as has been characterized previously (Leibiger et al. 2013). The 3T3-lacO cell line contains a modal number of three copies of chromosome 3 per cell (Fig. 2). One of these copies consists of two copies of chromosome 3 fused together at their q arms by an array of lac operator repeats, forming a structurally dicentric, but functionally monocentric chromosome (Soutoglou et al. 2007). One centromere on this chromosome is inactive, producing a single structure with an active centromere at one end of the chromosome and an array of lacO repeats at the center (Fig. 2).Fig. 1

Bottom Line: We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes.Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar.Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.

ABSTRACT
Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

Show MeSH
Related in: MedlinePlus